Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
Human herpesviruses are major causes of adverse health effects. Human cytomegalovirus (HCMV), for example, is a major cause of birth defects, transplantation failure, and devastating disease in immunocompromised individuals. Herpesviruses and other DNA viruses such as papillomaviruses are particularly difficult problems for humans, because they form life-long persistent infections. An additional discussion of viral infections, in particular human cytomegalovirus infections, can be found in U.S. Pat. Nos. 4,663,317, 4,782,065, 4,800,081, and 4,849,412, the contents of each of which are incorporated herein.
Although some drugs have been developed that are efficacious in treating these virus infections, drug-related toxicity and development of drug-resistant virus strains have compromised their impact on treatment of these virus infections. These findings indicate that new therapeutic approaches are needed for these infections.
Human cytomegalovirus infection is widespread among human populations, primarily as a subclinical persistent infection, although HCMV infection is a major cause of morbidity and mortality in several well-studied risk groups. Those most severely affected by HCMV infection include congenitally infected infants and individuals whose immune systems are compromised, particularly with HIV infection or immunosuppressive therapy for tissue transplantation (for reviews, see 8, 27, 59, 63). The clinical management of these infections is still problematic, even though several agents have been identified with potent antiviral activity for HCMV infection both in vitro and in vivo. Unfortunately, the toxicity associated with the long-term use of these drugs makes clinical management difficult, and drug resistant strains have emerged (for a review, see 54). Thus, there continues to be great interest in improving the understanding of the replication of HCMV with a view towards developing more effective approaches to control these infections.
HCMV replication seems to be closely associated with extensive modifications of cellular metabolism (reviewed in 4, 5), leading to a number of physiologic changes and activation of a large number of cellular genes (76). Initially, HCMV infection induces a series of cellular responses that in many ways resembles the immediate early events observed following activation of serum-arrested cells by serum growth factors (4). These events include: hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), yielding increased cellular levels of sn-1,2-diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3) (69); increased release of arachidonic acid and its metabolites (1,2); changes in Ca2+ homeostasis, including Ca2+ influx, release of Ca2+ stores, and an increase in intracellular free Ca2+ (51); transcriptional activation of cellular oncogenes c-fos, c-jun, and c-myc (11,12,13); and increased activity of the DNA-binding proteins NFκB, AP-1, and CREB (14). The signaling cascade induced by HCMV infection induces a robust mitogenic response. This is evidenced by the ability of HCMV to stimulate density-arrested cells, which are resistant to stimulation by serum growth factors, to enter the cell cycle (18). Recent results indicate that productive HCMV infection stimulates cell cycle progression in either serum- or density-arrested cells through late G1 phase to a point at or near the G1/S boundary (18,28,46). Closely associated with this limited traverse of the cell cycle is an increase in cyclin E/cyclin-dependent kinase 2 (Cdk2) activity (18) and hyperphosphorylation of pRb, releasing E2F (41). Activation of E2F, together with MYC, leads to an increase in the cellular levels of a large number of genes involved in nucleotide biosynthesis, priming the infected cell for DNA synthesis (e.g., 5,7).
Three HCMV-induced events appear to be necessary for activation of E kinase activity: 1) transcriptional activation of cyclin E (16), 2) translocation of Cdk2 from the cytoplasm to the nucleus (19), and 3) a substantial decrease in the nuclear levels of the cyclin kinase inhibitors (CKIs) p21cip1/waf1 (hereafter p21cip1) and p27kip1 (18). Activation of E kinase appears to be critical for efficient HCMV replication, since drugs that interfere with the activity of Cdk2 substantially reduce infectious yields (17). The precise mechanisms through which these virus-induced cellular modifications are achieved are poorly understood at this time.
p21cip1 is a potent inhibitor of Cdks (e.g., 37,38,71) and is a critical p53 downstream effector in the growth suppressive pathway (31). p21cip1 binds cyclin/Cdk complexes, thereby inhibiting the activity of Cdks, such as Cdk2, Cdk3, Cdk4, and Cdk6, and consequently inhibiting cell cycle progression. In addition, p21cip1 interacts with proliferating cell nuclear antigen (PCNA) (34) and gadd45 (42), affecting their function, e.g., interfering with DNA replication and repair (22,34,45,53,55,70,73). p21cip1 may also be involved in p53-mediated apoptosis (36) and in the control of cell senescence (50). Despite significant advances in the understanding of how p21cip1 exerts its biological effects and is transcriptionally regulated, there is only limited information available on how the stability of the p21cip1 protein is regulated under the physiologic conditions associated with disease and other forms of stress. Non-lysosomal cytoplasmic protease systems have been identified as important regulators of cell cycle progression (24,26,33,52,61,62). Two prominent cytoplasmic protease pathways have been identified—the ubiquitin-proteasome and calpain pathways. Many cell cycle regulatory proteins that are degraded at specific points in the cell cycle, e.g., cyclins A, B, and E, are substrates of the ubiquitin/proteasome pathway. It has also been reported that p21cip1 is subject to proteolysis by ubiquitin-mediated proteasome degradation (10,33,35,72).